Tissue engineering scaffolds promoting matrix protein production

ABSTRACT

The present invention provides tissue engineering scaffolds capable of inducing extracellular matrix production by a cell attached to the tissue engineering scaffolds, the tissue engineering scaffolds comprising: a scaffold; a polymer tether covalently coupled to the scaffold; and a TGF-β molecule that is covalently coupled to the polymer tether, wherein the TGF-β molecule is present at a concentration sufficient to elicit production of extracellular matrix by the cell attached to the tissue engineering scaffold without increasing cellular proliferation of the attached cell.

CROSS-REFERENCE TO RELATED APPLICATION

This Application is a divisional application of U.S. patent applicationSer. No. 09/935,168 entitled “Tissue Engineering Scaffolds PromotingMatrix Protein Production,” filed on Aug. 21, 2001, and claimingpriority to U.S. application Ser. No. 60/226,771, filed Aug. 21, 2000.

SEQUENCE LISTING

The Sequence Listing in this application is identical to thecomputer-readable copy of the Sequence Listing filed in U.S. patentapplication Ser. No. 09/935,168 entitled “Tissue Engineering ScaffoldsPromoting Matrix Protein Production,” filed on Aug. 21, 2001, and isincorporated herein by reference.

BACKGROUND

The present invention is generally in the field of improved compositionsfor tissue engineering, specifically scaffolds incorporating defineddensities of matrix-enhancing molecules for improving matrix proteinproduction of cells, without inducing excessive proliferation of thecells.

In fields where cell growth, maintenance, or production of exogenousfactors are important, such as in the field of tissue engineering, cellsare often grown on solid substrates or scaffolds which provide asuitable substrate for cell adhesion and growth. These scaffolds may bemade of natural or synthetic materials.

Biomaterials developed for tissue engineering and wound-healingapplications need to support adequate cell adhesion while being replacedby new tissue synthesized by those cells. In order to maintain propermechanical integrity of the tissue, the cells must generate sufficientextracellular matrix (ECM). Decreased ECM production by cells in tissueengineering scaffolds may lead to reduced structural integrity of thedeveloping tissue.

In order to optimally promote adhesion to such materials, researchershave investigated attachment of cell adhesion ligands, such as theArginine-Glycine-Aspartic acid (RGD) peptide, to surfaces ofbiomaterials (Massia & Hubbell, Anal. Biochem. 187:292-301 (1990); Hern& Hubbell, J. Biomed. Mater. Res. 39:266-76 (1998); Dee, et al. J.Biomed. Mater. Res. 40:371-77(1998); Tong & Shoichet, J. Biomed. Mater.Res. 42:85-95 (1998); Zhang, et al., Biomaterials 20:1213-20 (1999)).However, an increase in cell adhesion can adversely affect ECMproduction (Mann, et al., Biomaterials 20:2281-86 (1999)). In addition,there exists a substantial need to increase ECM production, even inunmodified scaffolds, as the proteins in the ECM largely determine themechanical properties of the resultant tissue and are often needed toreplace the functions of a biodegradable scaffold material. Themechanical properties of the resultant tissue are particularly importantin applications such as tissue engineered vascular grafts and orthopedictissue engineering wherein failure can occur due to poor mechanicalintegrity.

Researchers have also attached growth factors such as transforminggrowth factor (TGF) to a tissue engineering matrix via a polymerictether such as a polyethylene glycol. See WO 96/27,657, “Cell GrowthSubstrates with Tethered Cell Growth Effector Molecules.” There are anumber of references that TGF-β can be bound to or dispersed within asynthetic or natural polymeric carrier for controlled release of activegrowth factor. See, e.g., Schroeder-Tefft, et al., “Collagen and heparinmatrices for growth factor delivery,” Journal of Controlled Release49(2-3), 291-98 (1997); Nicoll, et al., “In vitro characterization oftransforming growth factor-beta-1-loaded composites of biodegradablepolymer and mesenchymal cells,” Cells and Materials 5(3), 231-44 (1995).EP 00/42,8541, “Collagen Wound Healing Matrices and Process for theirProduction” to Collagen Corporation; U.S. Pat. No. 6,013,853,“Continuous release polymeric implant carrier” issued to Athanasiou, etal. Additional references relate to the use of TGF-β in tissueengineering scaffolds to enhance cell or tissue growth or proliferation,particularly of bone. See EP 0616814, “Ceramic and Polymer-BasedCompositions for Controlled Release of Biologically Active TGF-β to BoneTissue, and Implants Using the Compositions,” by Bristol-Myers SquibbCompany.

However, none of these disclosures disclose how one can achieve enhancedproduction of extracellular matrix, while not increasing cellularproliferation.

It is therefore an object of the present invention to provide tissueengineering scaffolds which promote formation of ECM, to enhance theformation of tissue with good mechanical properties, on and within thetissue engineering scaffold, i.e., with little or no increase incellular proliferation.

SUMMARY

It has been found that matrix-enhancing molecules, such as TGF-β, can beconjugated to or immobilized on scaffolds to increase ECM production bycells. The matrix-enhancing molecule is conjugated to a polymer, such aspolyethylene glycol (PEG) monoacrylate, for attachment to a tissueengineering or cell growth scaffold, useful in not only tissueengineering but also for tissue regeneration and wound-healingapplications. The matrix-enhancing molecule retains activity afterattachment to the scaffold and causes cells growing in or on thescaffold to increase ECM production, even when the scaffold additionallycontains cell adhesion ligands. This increase in ECM production isbelieved to be due to an increase in gene expression, not cellproliferation. The increased ECM produced by the cells aids inmaintaining the integrity of the scaffold, particularly when thescaffold is degradable, either by hydrolysis or by enzymaticdegradation.

The examples demonstrate that matrix production by vascular smoothmuscle cells (SMCs) grown in a polymer scaffold, which was formed fromPEG hydrogels containing covalently bound adhesive ligands, wasincreased in the presence of 4×10⁻⁵ nmol TGF-β/mL tethered to thescaffold even when no TGF-β was present. At the same time, cellproliferation was not increased, which is advantageous since increasedproliferation of SMCs could lead to narrowing of a vessel lumen.Tethering TGF-β to the polymer scaffold resulted in a significantincrease in extracellular matrix production over the same amount ofsoluble TGF-β (see Example 1, FIG. 3). This is most likely due tointernalization of the soluble TGF-β by the cells or diffusion of thesoluble TGF-β from the hydrogels, making the TGF-β unavailable to thecells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of matrix production per cell by SMCs growing on glasssurfaces covalently coupled with RGDS (SEQ ID NO:1), VAPG (SEQ ID NO:2),KQAGDV (SEQ ID NO:3), or RGES (SEQ ID NO:4) with and without TGF-β inthe media. The “+” indicates that TGF-β was added to the media, whilethe “−” indicates that TGF-β was absent from the media.

FIG. 2 is a graph of matrix production per cell by SMCs growing on RGDS(SEQ ID NO:1)-modified glass surfaces with no TGF-β, soluble TGF-β oracryloyl-PEG-TGF-β at 4×10⁻⁵ nmol/mL in the hydrogel. The control iswith no TGF-β in the hydrogel.

FIG. 3 is a graph of hydroxyproline production per ng of DNA by SMCsgrowing in RGDS (SEQ ID NO:1)-containing hydrogels with no TGF-β,soluble TGF-β, or acryloyl-PEG-TGF-β at 4×10⁻⁵ nmol/mL in the hydrogel.The control is with no TGF-β in the hydrogel.

FIG. 4 is a graph of matrix production by SMCs growing on RGDS (SEQ IDNO:1)-modified glass surfaces, as percent of control, as a function ofTGF-β concentration (0, 1, and 5 ng/mL).

FIG. 5 is a graph of matrix production, as percent of control, byauricular chondrocytes growing on tissue culture polystyrene in thepresence of varying amounts of TGF-β (0, 0.1, 1, 5, 10, 25, and 100ng/mL) in the media. The control is with no TGF-β in the media.

FIG. 6 is a graph of cell number, as percent of control, by auricularchondrocytes growing on tissue culture polystyrene in the presence ofvarying amounts of TGF-β (0, 0.1, 1, 5, 10, 25, and 100 ng/mL) in themedia. The control is with no TGF-β in the media.

FIG. 7 is a graph of matrix production, as percent of control, byauricular chondrocytes and aortic smooth muscle cells growing on tissueculture polystyrene in the presence of 0 and 50 μg/mL ascorbic acid inthe media. The control is with no ascorbic acid in the media.

FIG. 8 is a graph of cell number, as percent of control, by auricularchondrocytes and aortic smooth muscle cells growing on tissue culturepolystyrene in the presence of 0 and 50 μg/mL ascorbic acid in themedia. The control is with no ascorbic acid in the media.

DESCRIPTION

Tissue engineering is performed using a scaffold material that allowsfor attachment of cells. The scaffold material contains amatrix-enhancing molecule. As described herein, the matrix-enhancingmolecule should promote the production of extracellular matrix proteins,but should not promote cell proliferation.

Scaffold Materials

In the preferred embodiment, the scaffold is formed of synthetic ornatural polymers, although other materials such as hydroxyapatite,silicone, and other inorganic materials can be used. The scaffold may bebiodegradable or non-biodegradable.

There are a number of biocompatible polymers, both degradable andnon-degradable. Representative synthetic non-biodegradable polymersinclude ethylene vinyl acetate and poly(meth)acrylate. Representativebiodegradable polymers include polyhydroxyacids such as polylactic acidand polyglycolic acid, polyanhydrides, polyorthoesters, and copolymersthereof. Natural polymers include collagen, hyaluronic acid, andalbumin.

A preferred material is a hydrogel. A particularly preferredhydrogel-forming material is a polyethylene glycol-diacrylate polymer,which is photopolymerized. Other hydrogel materials include calciumalginate and certain other polymers that can form ionic hydrogels thatare malleable and can be used to encapsulate cells.

Formation of Scaffolds

Scaffolds can be formed in situ or in vitro. In a preferred embodimentfor formation of joint linings, the scaffold material is sprayed in adilute solution onto the joint, then polymerized, so that the polymerforms a hydrogel coating bonded onto the surface of the joint tissues.Cells can be dispersed within the polymer, or seeded onto the polymericmatrix. Scaffolds may also be formed of fibers of polymer, woven ornon-woven into meshes that can be used to support cell attachment andgrowth. These scaffolds can be formed by casting, weaving, saltleaching, spinning, or molding. In still another embodiment, scaffoldscan be formed using molds formed by micromachining and photolithographictechniques, where the cells can be seeded into the scaffold while in themolds or after removal of the scaffold. In a preferred embodiment, aliquid cell-polymer solution is placed in a mold and photopolymerized,converting the liquid to a hydrogel with the cells seeded within thehydrogel.

The scaffolds can be seeded at the time of or before implantation at asite where tissue is desired. Meshes should preferably be sufficientlyopen to allow free diffusion of nutrients and gases throughout thescaffold.

Matrix-Enhancing Molecules

Matrix-enhancing molecules which promote increased production of ECM canbe attached to the scaffold material to induce production of matrixproteins, such as glycoproteins, elastin, and collagen, withoutsubstantially increasing cell proliferation. These matrix-enhancingmolecules include TGF-β, angiotensin II, insulin-like growth factors,and ascorbic acid.

TGF-β is known to increase production of extracellular matrix proteinsby vascular SMCs growing in culture (Amento, et al., Arterioscler.Thromb. 11:1223-30 (1991); Lawrence, et al., J. Biol. Chem. 269:9603-09(1994); Plenz, et al., Atherosclerosis 144:25-32 (1999)). TGF-β, throughproduction by SMCs naturally during vessel injury or by gene transfer,can also increase ECM production by SMCs in vivo (Majesky, et al., J.Clin. Invest. 88:904-10 (1991); Nabel, et al., Proc. Natl. Acad. Sci.USA 90:10759-63 (1993)). Cultured fibroblasts have also been shown toincrease collagen synthesis (Clark, et al., J. Cell Sci. 108:1251-61(1995); Eickelberg, et al., Am. J. Physiol. 276:L814-24 (1999)) andproteoglycan synthesis (Heimer, et al., J. Mol. Cell Cardiol. 27:2191-98(1995)) in the presence of TGF-β. Further, topical delivery of TGF-β(Puolakkainen, et al., J. Surg. Res. 58:321-29 (1995)) and delivery toTGF-β through a collagen scaffold (Pandit, et al., J. Invest. Surg.12:89-100 (1999)) have been shown to enhance wound healing.

All of the above-referenced studies have examined effects in thepresence of soluble TGF-β. As demonstrated by the following examples, ithas now been shown that tethered TGF-β can also be used to induceformation of ECM by cells, including cells such as smooth muscle cellsand chondrocytes.

Tethers

For the matrix-enhancing molecules to induce formation of ECM, it isnecessary for the molecule to be tethered to the scaffold by a tether.These tethers have a molecular weight of preferably between about 200and 10,000, most preferably between about 2,000 and 6,000. The tether ispreferably a linear polymer, such as polyethylene glycol. Thematrix-enhancing molecule may be coupled to the tether or, for thatmatter, to the scaffold material, by any method known to those of skillin the art, preferably covalently coupled using a reagent such asN-hydroxysuccinimide, carbodiimide, diisocyanate, carbonyldiimidazole,or tosyl chloride.

Density of Matrix-Enhancing Materials

The density of the matrix-enhancing materials is important in elicitingECM production with little or no cellular proliferation. The amount ofECM production that is most desirable is that which results in formationof tissue with good mechanical properties, on and within the tissueengineering scaffold. The optimal density will depend on the type ofcells to be attached to the scaffold. In the case of TGF-β, optimalconcentrations to induce ECM production is in the range of between oneand five ng TGF-β/mL for aortic smooth muscle cells and between 5 and100 ng TGF-β/mL for auricular chondrocytes, which is equivalent tobetween 4×10⁻⁶ and 4×10⁻³ nmol/mL.

Source of Cells

Cells can be obtained directly from a donor, from a culture of cellsfrom a donor, or from established cell culture lines. In the preferredembodiment, cells of the same species and, preferably having the same ora similar immunological profile, are obtained by biopsy, either from thepatient or a close relative, which can then be grown in culture usingstandard conditions. If cells that are likely to elicit an immunereaction are used, such as human muscle cells from an immunologicallydistinct individual, then the recipient can be immunosuppressed asneeded, for example, using a schedule of steroids and otherimmunosuppressant drugs, such as cyclosporine.

In the preferred embodiments, cells are obtained directly from a donor,washed and implanted directly in combination with the polymericmaterial. The cells are cultured using techniques known to those skilledin the art of tissue culture. Cells obtained by biopsy are harvested andcultured, passaging as necessary to remove contaminating cells.

Preferred cells for formation of vascular tissue include smooth musclecells, endothelial cells, and fibroblasts. Preferred cells for formationof connective tissue include chondrocytes, fibroblasts, and other typesof cells that differentiate into bone or cartilage.

Methods of Using the Scaffolds

The scaffolds are used to produce new tissue, such as vascular tissue,cartilage, tendons, and ligaments. The scaffold is typically seeded withthe cells; the cells are cultured; and then the scaffold implanted.Alternatively, as noted above, the scaffold is sprayed into or onto asite such as a joint lining, and seeded with cells, and then the site isclosed surgically. Liquid polymer-cell suspensions can also be injectedinto a site, such as within a joint, where the material may bepolymerized.

Applications include the repair and/or replacement of organs or tissues,such as blood vessels, cartilage, joint linings, tendons, or ligaments,or the creation of tissue for use as “bulking agents,” which aretypically used to block openings or lumens, or to shift adjacent tissue,as in treatment of reflux.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLES Example 1 Comparison of Effect of Soluble and Bound TGF-β inCombination with PEG-Diacrylate Hydrogel

It was determined whether TGF-β can counteract the decrease in ECMsynthesis caused by immobilized cell adhesion ligands. SMCs were grownon both peptide-modified glass substrates and in hydrogels containingtethered cell adhesion ligands. Further, TGF-β was covalently tetheredto a polymer scaffold and shown that it retains its ability to increaseECM production.

Materials & Methods

Cell Maintenance

Chemicals were obtained from Sigma Chemical Co. (St. Louis, Mo.) unlessotherwise stated. SMCs from the thoracic aorta of Wistar-Kyoto rats wereisolated and characterized as previously described by Scott-Burden, etal., Hypertension 13:295-05 (1989). Human aortic smooth muscle cells(HASMCs) were obtained from Clonetics (San Diego, Calif.). Both SMCs andHASMCs were maintained on Minimal Essential Medium Eagle supplementedwith 10% fetal bovine serum (FBS; Bio Whittaker, Walkersville, Md.), 2mM L-glutamine, 500 units penicillin and 100 mg/L streptomycin (MEM).Cells were incubated at 37° C. in a 5% CO₂ environment.

Surface Modification

Peptides used in this study were RGDS (SEQ ID NO:1), VAPG (SEQ ID NO:2),and KQAGDV (SEQ ID NO:3) (Research Genetics, Huntsville, Ala.). RGES(SEQ ID NO:4) was used as a non-adhesive control peptide. The peptideswere acetylated and coupled to aminophase glass slides as previouslydescribed by Mann, et al., Biomaterials 20:2281-86 (1999). Briefly,aminophase slides were prepared by incubating glass slides with3-aminopropyltriethoxysilane in dry acetone at 37° C. overnight.Acetylated peptides were then coupled to the slides using1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDAC) chemistry. Slideswere sterilized under UV light overnight prior to use.

Preparation of Acryloyl-PEG-TGF-β

TGF-β was conjugated to polyethylene glycol (PEG) by reacting TGF-β withacryloyl-PEG-N-hydroxysuccinimide (acryloyl-PEG-NHS, 3,400 Da;Shearwater Polymers, Huntsville, Ala.) in 50 mM TRIS buffer (pH 8.5) for2 hours. The mixture was then lyophilized and stored frozen. Gelpermeation chromatography equipped with UV/Vis (260 nm) and evaporativelight scattering detectors was used to analyze the resultingacryloyl-PEG-TGF-β and PEG standards (Polymer Laboratories, Amherst,Mass.).

Evaluation of Matrix Protein Production on Surfaces

Matrix protein production was evaluated as previously described by Mann(1999). Suspensions of SMCs were prepared in MEM supplemented with 5μg/mL ascorbic acid at a concentration of 40,000 cells/mL. For samplesreceiving TGF-β, 0.04 pmol/L (1 ng/mL) unmodified TGF-β or 0.04 pmol/mLacryloyl-PEG-TGF-β was added to the media. Cell suspensions to be usedfor measurement of ECM protein production were also supplemented with 1μCi/mL ³H-glycine (40 Ci/mmol). The glass slides were attached toFlexiPerm strips in QuadriPerm Cell Culture Vessels (Heraeus, Osterodeam Harz, Germany) to create eight wells (1.11×0.79×0.79 cm) on eachslide. Four of the wells on each slide were utilized to measure ECMproduction, while the remaining four wells were utilized for cell numberdetermination and were cultured in the absence of ³H-glycine. Cellnumber was determined after 2 days of culture by preparing single cellsuspensions using trypsin and counting cells using a Coulter counter(Multisizer #0646, Coulter Electronics, Hialeah, Fla.).

To evaluate synthesis of ECM proteins, the cell growth media wassupplemented with ³H-glycine, as described above. Two days following³H-glycine addition, cells were removed non-enzymatically with 25 mMammonium hydroxide, then rinsed with 70% ethanol and dried. This processleaves intact the ECM elaborated by cells during culture (Jones, et al.,Proc. Natl. Acad. Sci. USA 76:353-57 (1979)). Sequential enzymedigestion was used as previously described by Mann et al. (1999) todetermine the gross composition of the ECM proteins. Briefly, the ECMwas digested first with trypsin, followed by elastase, and thencollagenase. After the final enzymatic digestion, any material remainingon the substrate was dissolved by incubation with 1 N NaOH. Aliquotswere taken from each step of the digest for scintillation counting.

Preparation of Acryloyl-PEG-RGDS (SEQ ID NO: 1)

RGDS (SEQ ID NO:1) was conjugated to acryloyl-PEG-NHS in the same manneras TGF-β.

Preparation of PEG-Diacrylate

PEG-diacrylate was prepared by combining 0.1 mmol/mL dry PEG (6,000 Da;Fluka, Milwaukee, Wis.), 0.4 mmol/mL acryloyl chloride, and 0.2 mmol/mLtriethylamine in anhydrous dichloromethane and stirring under argonovernight. The resulting PEG-diacrylate was then precipitated withether, filtered, and dried in a vacuum oven.

Preparation of Hydrogels

Hydrogels were prepared by combining 0.4 g/mL PEG-diacrylate, 1.4μmol/mL acryloyl-PEG-RGDS (SEQ ID NO:1), and 0.3 mmol/mL triethanolaminein 10 mM HEPES-buffered saline (pH 7.4, HBS). This aqueous polymersolution was sterilized by filtration (0.8 μm prefilter and 0.2 μmfilter) and added to an equal volume of a suspension of SMCs at 2×10⁶cells/mL, such that the resulting polymer-cell solution contained 1×10⁶cells/mL. For hydrogels containing TGF-β, 0.04 pmol/mL (1 ng/mL)unmodified TGF-β or 0.04 pmol/mL acryloyl-PEG-TGF-β was added to thepolymer-cell solution. Then, 40 μl of 2,2-dimethyl-2-phenyl-acetophenonein N-vinylpyrrolidone (600 mg/mL) was added, and 0.25 mL of the solutionwas placed in a disk-shaped mold (20 mm diameter, 2 mm thickness). Thisliquid polymer-cell solution was then exposed to UV light (365 nm, 10mW/cm²) for 20 seconds to convert the liquid polymer-cell solution to ahydrogel with homogeneously seeded cells. Hydrogels were incubated inMEM containing 10% FBS for 7 days at 37° C. with 5% CO₂. Media waschanged every 3 days.

DNA and Hydroxyproline Determination in Hydrogels

After 7 days of culture, hydrogels were removed from the culture media,weighed, and digested with 1 mL 0.1 N NaOH overnight at 37° C. Digestedhydrogels were then neutralized with 1 mL 0.1 N HCl. DNA content of thedigested, neutralized hydrogels was determined using a fluorescent DNAbinding dye, Hoechst 33258 (Molecular Probes, Eugene, Oreg.).Fluorescence of the samples was determined using a fluorometer(VersaFluor, Bio-Rad Laboratories, Hercules, Calif.) with excitationfilter at 360 nm and emission filter at 460 nm, and compared tofluorescence of calf thymus DNA standards.

Hydroxyproline concentration was determined by oxidation with chloramineT (ICN Biomedicals, Aurora, Ohio) and development withp-dimethylaminobenzaldehyde (ICN Biomedicals) (Woessner, et al., Arch.Biochem. Biophys. 93:440-47 (1961)). Hydroxyproline is a marker forcollagen production, and thus was used as an indication of matrixsynthesis.

Mechanical Testing of Hydrogels

Hydrogels were prepared as described above, except that 2 mL of theliquid polymer-cell solution was placed in a 3 mm thick rectangular mold(42 mm×14 mm). For this experiment, HASMCs were used at a final celldensity of 3.5×10⁵ cells/mL. Hydrogels contained either no TGF-β or 0.04pmol/mL acryloyl-PEG-TGF-β.

Following photopolymerization, the hydrogels were placed in QuadriPermCell Culture Vessels with 10 mL media containing 10% FBS and incubatedat 37° C. with 5% CO₂. Media was changed every 3 days. After 7 days ofculture, the hydrogels were cut into 3 sections (14 mm×14 mm), andmechanical testing was performed using a Vitrodyne V-1000 UniversalTester (Chatillon, Greensboro, N.C.) at a strain rate of 100 μm/s usinga 150 g loading cell.

Statistical Analysis

Data sets were compared using two-tailed, unpaired t-tests. P-Valuesless than 0.05 were considered to be significant.

Results

The goal of the current study was to determine whether TGF-β can enhancethe rate of ECM synthesis by cells grown on or in biomaterials,particularly materials that have been modified with cell adhesionligands. FIG. 1 shows the matrix protein production on a per cell basisfor cells grown with either no TGF-β or 0.04 pmol/mL (1 ng/mL) solubleTGF-β in the media. With no TGF-β in the media, more matrix was producedby cells growing on the non-adhesive control, RGES (SEQ ID NO:4), thanon the adhesive peptides. When TGF-β was added to the media, matrixproduction increased on the adhesive surfaces over that produced when noTGF-β was added (224% increase on RGDS (SEQ ID NO:1) surfaces, 20%increase on VAPG (SEQ ID NO:2) surfaces, 104% increase on KQAGDV (SEQ IDNO:3) surfaces). Matrix production on the RGDS (SEQ ID NO:1) and KQAGDV(SEQ ID NO:3) modified surfaces in the presence of TGF-β increased overthat seen with the non-adhesive control.

Cells seeded onto RGDS (SEQ ID NO:1)-modified glass surfaces were alsogrown in the presence of 0.04 pmol/mL acryloyl-PEG-TGF-β to determine ifTGF-β could be covalently bound to a polymer (covalently attached to asoluble polymer chain but not tethered to a three-dimensional structure)and retain its ability to increase ECM production. FIG. 2 shows thematrix production by cells grown with no TGF-β, soluble TGF-β, oracryloyl-PEG-TGF-β in the media. SMCs produced greater amounts of matrixin the presence of either soluble or polymer-conjugated TGF-β over thatproduced in the absence of TGF-β. However, less matrix was produced whenpolymer-conjugated TGF-β was used than when unmodified TGF-β was used.

SMCs were then homogeneously seeded into polyethylene glycol (PEG)hydrogels containing covalently tethered RGDS (SEQ ID NO:1). Thehydrogels contained either no TGF-β, unmodified (soluble) TGF-β, orPEG-tethered TGF-β. In these photopolymerized hydrogels, the tetheredpeptides of TGF-β are covalently bound to the hydrogel structure via ahighly flexible PEG chain. This gives the tethered moietiesconformational freedom to interact with their receptors while causingthem to be retained in the hydrogel material. After 7 days of culture,the hydrogels were digested and assayed for DNA and hydroxyproline.Since hydroxyproline is a marker for collagen, it is an indication ofhow much extracellular matrix has been produced.

The results for cells grown in the presence of 0.04 pmol/mL of TGF-β arepresented in FIG. 3. More hydroxyproline, and thus more collagen, wasproduced by SMCs grown in the presence of either soluble or tetheredTGF-β than when no TGFβ was present. Additionally, significantly morehydroxyproline was produced when TGF-β was tethered onto the hydrogelsthan when soluble TGF-β was used.

Additionally, the mechanical properties of hydrogels made with andwithout TGF-β were examined. Young's modulus, a measure of the stiffnessof the scaffold, was significantly higher when TGF-β was tethered to thescaffolds than when no TGF-β was used (66.6±3.7 kPa with tethered TGF-βversus 58.5±1.8 kPa with no TGF-β, p=0.03).

Example 2 Dose Response of Aortic Smooth Muscle Cells to TGF-β

Aortic smooth muscle cells were grown on aminophase glass that had 0.5nmol/cm² RGDS (SEQ ID NO:1) covalently coupled to the glass. TGF-β wasadded to the media at 0, 1, or 5 ng/mL (0, 4×10⁻⁵, 2×10⁻⁴ nmol/mL). ECMprotein production by the cells over a 2-day time period was determinedby examining the amount of ³H-glycine incorporated into the ECMelaborated by the cells.

As seen in FIG. 4, ECM protein production per cell (% of control) wasincreased when TGF-β was added to the media at both 1 and 5 ng/mL.Further, cell numbers did not increase over the 2 days, despite changesin matrix production per cell, indicating that the presence of TGF-β didnot increase proliferation of the SMCs, as seen in the hydrogels.

Example 3 Dose Response of Auricular Chondrocytes to TGF-β

Auricular chondrocytes were grown on tissue culture polystyrene withvarying amounts of TGF-β added to the media: 0, 1, 5, 10, 25, or 100ng/mL. ECM protein production by the cells over a 2-day time period wasdetermined by examining the amount of ³H-glycine incorporated into theECM elaborated by the cells.

As seen in FIG. 5, ECM protein production per cell was increased whenTGF-β was added to the media at concentrations above 1 ng/mL, with anoptimal concentration of 25 ng/mL (1×10⁻³ nmol/mL). Further, cellnumbers did not increase over the 2 days (see FIG. 6), despite changesin matrix production per cell, indicating that the presence of TGF-β didnot increase proliferation of the chondrocytes.

Example 4 Increase in Matrix Production in the Presence of Ascorbic Acid

Aortic smooth muscle cells and auricular chondrocytes were grown ontissue culture polystyrene with and without 50 μg/mL ascorbic acid addedto the media. ECM protein production by the cells over a 2-day timeperiod was determined by examining the amount of ³H-glycine incorporatedinto the ECM elaborated by the cells.

As seen in FIG. 7, ECM protein production per cell was increased in thepresence of ascorbic acid for both SMCs (light gray) and chondrocytes(dark gray). Further, cell numbers did not increase over the 2 days(FIG. 8), despite changes in matrix production per cell, indicating thatthe presence of ascorbic acid did not increase proliferation of thesmooth muscle cells (light gray) or the chondrocytes (dark gray).

Modifications and variations of these scaffolds and method forpreparation and use thereof will be obvious to those skilled in the artand are intended to be encompassed by the following claims.

1. A tissue engineering scaffold capable of inducing extracellularmatrix production by a cell attached to the tissue engineering scaffoldwithout increasing cellular proliferation of the attached cell, thetissue engineering scaffold comprising: a scaffold; a polymer tethercovalently coupled to the scaffold; and a TGF-β molecule that iscovalently coupled to the polymer tether, wherein the TGF-β molecule ispresent at a concentration sufficient to elicit production ofextracellular matrix by the cell attached to the tissue engineeringscaffold without increasing cellular proliferation of the attached cell.2. The tissue engineering scaffold of claim 1 further comprising a cellattached to the tissue engineering scaffold.
 3. The tissue engineeringscaffold of claim 2 wherein the cell is attached to the tissueengineering scaffold by constraining the cell within the scaffold. 4.The tissue engineering scaffold of claim 3 wherein the scaffold is ahydrogel.
 5. The tissue engineering scaffold of claim 2 wherein the cellis selected from the group consisting of smooth muscle cells,endothelial cells, fibroblasts, chondrocytes, and combinations thereof.6. The tissue engineering scaffold of claim 1 wherein the polymer tetherhas a molecular weight of between about 200 and about 10,000.
 7. Thetissue engineering scaffold of claim 1 wherein the tether has amolecular weight of between about 2,000 and about 6,000.
 8. The tissueengineering scaffold of claim 1 wherein the scaffold is formed from abiocompatible polymer selected from the group consisting of a syntheticpolymer, a natural polymer, an inorganic material, and a combinationthereof.
 9. The tissue engineering scaffold of claim 1 wherein thescaffold is formed from a biocompatible natural polymer, and wherein thebiocompatible natural polymer is selected from the group consisting of acollagen, a hyaluronic acid, an albumin, and a combination thereof. 10.The tissue engineering scaffold of claim 1 wherein the scaffold isformed from a biocompatible inorganic material, and wherein thebiocompatible inorganic material is selected from the group consistingof a hydroxyapatite, a silicone, and a combination thereof.
 11. Thetissue engineering scaffold of claim 1 wherein the scaffold is formedfrom a biocompatible synthetic polymer, and wherein the biocompatiblesynthetic polymer is selected from the group consisting of an ethylenevinyl acetate, a poly(meth)acrylate, and a combination thereof.
 12. Thetissue engineering scaffold of claim 1 wherein the scaffold is formedfrom a biocompatible biodegradable polymer.
 13. The tissue engineeringscaffold of claim 1 wherein the scaffold is formed from a biocompatiblebiodegradable polymer, and wherein the biocompatible biodegradablepolymer is selected from the group consisting of a polyhydroxyacid, apolylactic acid, a polyglycolic acid, a polyanhydride, a polyorthoester,and a combination thereof.
 14. The tissue engineering scaffold of claim1 wherein the scaffold is formed from a biocompatible polymer that isnot biodegradable.
 15. The tissue engineering scaffold of claim 1wherein the scaffold is formed from a biocompatible polymer that is ahydrogel.
 16. The tissue engineering scaffold of claim 1 wherein thescaffold is formed from a biocompatible polymer that is a polyethyleneglycol-diacrylate polymer hydrogel.
 17. The tissue engineering scaffoldof claim 1 wherein the scaffold is formed from a biocompatible polymerthat is an alginate hydrogel.
 18. The tissue engineering scaffold ofclaim 1 wherein the scaffold is formed from a biocompatible polymer thatis a malleable, ionic hydrogel.
 19. A tissue engineering scaffoldcapable of inducing extracellular matrix production by a cell attachedto the tissue engineering scaffold without increasing cellularproliferation of the attached cell, the tissue engineering scaffoldcomprising: a scaffold; a polymer tether covalently coupled to thescaffold; and a TGF-β molecule that is covalently coupled to the polymertether, wherein the TGF-β molecule is present at a concentration in therange of from about 4×10⁻⁶ to about 4×10⁻³ nmol/mL.
 20. The tissueengineering scaffold of claim 19, further comprising a cell attached tothe tissue engineering scaffold.
 21. A tissue engineering scaffoldcapable of inducing extracellular matrix production by a cell attachedto the tissue engineering scaffold without increasing cellularproliferation of the attached cell, the tissue engineering scaffoldcomprising: a scaffold, at least a portion of the scaffold comprising ahydrogel; a polymer tether covalently coupled to the scaffold; and aTGF-β molecule that is covalently coupled to the polymer tether, whereinthe TGF-β molecule is present at a concentration in the range of fromabout 4×10⁻⁶ to about 4×10⁻³ nmol/mL.
 22. The tissue engineeringscaffold of claim 21, further comprising a cell attached to the tissueengineering scaffold.